Influence of sperm viability on uterine leukocyte chemotaxis

Abstract

The immune system can differentiate live from dead microorganisms, generating a response as necessary; there has been limited study of equine sperm. We aimed to compare the uterine cellular immune response to artificial insemination (AI) with live versus dead equine sperm. Twenty mares in estrus (follicles ≥ 35 mm, uterine edema), without intrauterine fluid or polymorphonuclear leukocytes (PMNs) were selected from a population of horses sent to an abattoir during the breeding season. Their average age was 10.8 ± 0.9 years (range 6 - 20), body condition score > 3 (scale 1 - 5), and they were clinically healthy. Mares were randomly allocated into two groups. Frozen semen, a pool from two stallions, was inseminated into the uterine body: either LIVE (n = 10), 3.0 mL of 900 × 106 sperm (≥ 35% progressive motility), or DEAD (n = 10), 3.0 mL of 900 × 106 sperm killed by snap freezing in liquid nitrogen. Four hours post AI, mares were slaughtered; ex vivo uteri were subjected to a low-volume lavage (LVL) with 50 mL of PBS and three endometrial tissue samples were collected (both uterine horns and uterine body). The fluid recovered was analyzed in a Neubauer chamber. Endometrial samples were processed for histologicalevaluation using bright-field microscopy. For each mare, the three uterine samples were pooled for analysis. The number of PMNs in the stratum compactum (SC) and spongiosum (SS) was counted in 5 fields per uterine sample at 400x magnification, totaling 15 fields/stratum/mare. Groups were considered independent factors. Leukocyte concentrations in the LVL and PMN presence in both strata were considered dependent factors. Leukocyte concentrations in the LVL were analyzed with a Mann-Whitney test (median ± IQR) and the number of PMNs per field of each stratum using a t-test (mean ± SEM). The significance level was defined as p ≤ 0.05. Mares in the DEAD group presented a lower concentration of leukocytes in the LVL than in the LIVE group (33.2 × 106/mL ± 62.6 versus 63.2 × 106/mL ± 56.5; p = 0.05). Correspondingly, the mean PMN number per field in the SC (50.5 ± 5.2/field versus 69.0 ± 5.3/field; p = 0.02) and SS (32.8 ± 6.2/field versus 54.4 ± 5.7/field; p = 0.02) of mares in the DEAD group were lower than mares in the LIVE group. In conclusion, dead sperm elicited a less intense inflammatory response 4 hours after AI compared to live sperm, suggesting that as with bacteria, the immune system may differentiate sperm according to viability.